The ion activity measurements conducted in the assays described in U.S. Pat. No. 4,053,381, issued Oct. 11, 1977, are obtained by using a differential potential measurement on two substantially identical strips of an ion-selective electrode (hereinafter, "ISE"), responsive to a particular ion. As used herein, "dry-operative" refers to the ability of the ISE to be used within minutes, without delays for equilibration with H.sub.2 O. This is a property that appears to be peculiar to the ISE's described in the aforesaid patent. Aliquots of a sample, e.g., biological fluid, and a so-called "reference liquid", are both deposited. Each aliquot is deposited so that it contacts one of the two ISE's and one end of an ion bridge that allows the sample and reference solution to make liquid, and thus electrical, contact between the ends of the bridge. The voltage difference between the two electrodes is noted, and this voltage difference is converted by a calibration technique to ion concentration for that particular ion.
The reference liquid has several critical functions. For any given ion test, and therefore, for any given kind of ISE, that liquid must set a stable electrode potential (called "poising" the electrode) that is essentially constant from ISE to ISE for that type. That is, the method of calibration is set up on the assumption that such potential will be essentially constant within a manufacturing lot. A typical manufacturing lot will contain up to several hundred thousand ISE's, and the goal is for each ISE within the lot to be identical if tested with similar fluids.
In addition, it must reduce any variation in liquid junction potential when the reference liquid contacts the sample. For this reason, the reference liquid is analogous, not to calibrators, but to salt-bridge fluids such as saturated KCl, used in single or double junction reference electrodes such as the saturated calomel electrode.
Prior to this invention, commercial reference liquids for systems, such as that provided by Eastman Kodak Company for use with the Ektachem 400.TM. or 700.TM. Analyzer of Eastman Kodak Company, have been successful in most respects. However, they lacked equal ion transference that provides sufficient equal solution mobility for both the cations and anions. As a result, the ion transference at the liquid junction with the patient sample lacked the ability to swamp out, or dominate, a bias that is otherwise created when the sample ion content changes drastically (such as from patient to patient). What was observed in practice is that part of the change in the patient's ISE potential was created by a change in liquid junction potential rather than solely by a true change in ion activity. Because such changes in junction potential are not a true measure of the changed ion content, there is thus introduced a random bias. Such a bias makes proper operation difficult, since it is not amenable to correction by calibration.
For obvious reasons of simplicity, such Eastman Kodak Company reference liquid has a single composition that contains all the ions necessary for use in all the ion tests of interest, namely K.sup..sym., Na.sup..sym., Cl.sup..crclbar. and CO.sub.2. That is, it is obviously too cumbersome to switch to a different reference fluid just because a different ion is being tested in the next ISE test. Thus, a typical composition for such a conventional commercial reference liquid is a solution comprising Na.sup..sym., Cl.sup..crclbar., small amounts of K.sup..sym. (0.0045M), acetate.sup..crclbar., and HCO.sub.3.sup..crclbar., with a total ionic strength of 0.15M. The HCO.sub.3.sup..crclbar. anion is used to poise the ISE for the analysis of CO.sub.2. The 0.0045M amount of K.sup..sym. is less than conventionally considered adequate (1M) to provide equal ion solution transference.
It has also been known prior to this invention, that certain ions do provide equitransference, or sufficient equal solution mobility, such as will swamp out the junction potential noted above created by varying concentrations of the ion of choice. Thus, for the chloride anion, it has been known that the following cations have approximate equal solution mobility: potassium, ammonium, cesium and rubidium. This is apparent from the approximately equal equivalent ionic conductivity values given for these and for Cl in, e.g., pages D-171 and 172 of the CRC Handbook of Chemistry & Physics, 65th Ed., 1984.* (These have not been described, however, for use with an anion used to poise an electrode to assay for CO.sub.2.) Because the commercial reference liquid included the bicarbonate anion to allow usage with the CO.sub.2 ISE, it is readily evident that the ammonium cation is unacceptable in such a combination. That is, there is no pH that will keep the bicarbonate from converting into CO.sub.2 gas that escapes from the liquid (which occurs at a pH .ltoreq.8.0) and at the same time keeps the ammonium from converting into NH.sub.3 that escapes from the liquid (at a pH .gtoreq.8.0). FNT *Tl.sup..sym. also is shown with sufficient solution mobility, but it is known to be unacceptable because of its toxicity and expense.
It has also been assumed prior to this invention that anions other than Cl.sup..crclbar. can be used in an equitransferant salt to overcome a liquid junction potential.